BRAZILIAN SOIL
Juliana Alves Macedo1∗∗∗∗, Lara Durães Sette2, Hélia Harumi Sato1
Capítulo baseado nos trabalhos: Comunicação breve aceita para publicação na Food Bioprocess Technology e no artigo completo submetido para publicação no
Journal of Food Biochemistry.
∗ Corresponding author: Tel.: + 55 (19) 3521-2175; Fax +55 (19) 3289-1513. 1
Food Science Department, Faculty of Food Engineering, UNIVERSIDADE ESTADUAL DE CAMPINAS (UNICAMP) P.O. Box 6121, CEP 13083-862, SP, Brazil.
2
Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI), Microbial Resource Division, CPQBA/UNICAMP, P.O. Box 6171, CEP 13081-970, SP, Brazil.
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ABSTRACT
A new microbial transglutaminase (MTGase or MTG, EC 2.3.2.13) from a Streptomyces sp. strain isolated from Brazilian soil samples was purified and characterized. Enzyme purification was fast and simple, consisting of two successive chromatographies on Sephadex G-75 columns with yields of 48% and 17% respectively. The protein purification was successfully achieved to electrophoretical homogeneity on SDS-PAGE. The molecular mass of the MTGase was estimated to be about 45 kDa. The enzyme from Streptomyces sp., in both crude and pure forms, exhibited optimal activity in the 6.0-6.5 pH range and at 35-40°C. The results for the commercial enzyme were the same. A second maximum of activity was observed at pH 10.0 with both the crude Streptomyces sp. enzyme and the commercial enzyme. This interesting fact has not been reported in the literature previously. All of the enzymes tested were stable over the pH range from 4.5 to 8.0 and up to 45°C. The decline in activity of the commercial transglutaminase above 45°C and pH 8.0 was more gradual. The activities of all the MTGase samples were independent of Ca+2 concentration, but they were elevated in the presence of K+, Ba2+, and Co2+ and inhibited by Cu2+ and Hg2+, which suggests the presence of a thiol group in the MTG’s active site. The purified enzyme presented a Km of 6.37 mM and a Vmax of 1.7
U/mL, while the crude enzyme demonstrated a Km of 6.52 mM and a Vmax of 1.35 U/mL.
Keywords: transglutaminase, Streptomyces sp. CBMAI 837, enzyme purification, Sephadex G-75, biochemical characterization.
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INTRODUÇÃO
Transglutaminase (EC 2.3.2.13; protein-glutamine γ-glutaminyltranferase) is an enzyme that catalyses an acyl transfer reaction using peptide-bond glutamine residues as acyl donors and several primary amines as acceptors. When the ε-amino groups of the protein-bond lysyl residues are present as acyl receptors, this enzyme is capable of forming intra-and intermolecular ε-(γ-Glu)-Lys isopeptide bonds (Soares et al., 2003).
The covalent cross-links between a number of proteins and peptides introduced by transglutaminase promote modification of the functional properties of food proteins (Yokoyama et al., 2004). Therefore, transglutaminase-catalyzed reactions may be widely used by food-processing industries in the creation of new products, modification of the viscosity, alterations in the emulsifying and foaming properties, and improvement of the product‘s nutritional value (Zhu et al., 2004; Kwan & Easa, 1995).
Transglutaminases are found in mammalian tissues, plasma, fish, and plants (Pasternack et al., 1998). The mammalian enzymes are Ca+2-dependent. However, the relatively small quantities obtained and the necessary complex separation and purification procedures led to the search for alternative microbiological sources such as Candida albicans, Bacillus circulans, Physarum polycephalum and many Streptomyces species (Yokoyama et al., 2004; Zhu et al., 1995; Herrera et al., 1995; Souza et al., 2006; Klein et al., 1992).Ando et al. (1989) first reported that strains from the genus Streptoverticillium screened from several thousand microorganisms had the ability to produce transglutaminase using the hydroxamate assay. These microorganisms excreted the enzyme, and one of them, which was classified as a variant of Streptoverticillium
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mobaraensis, had high activity (Cui, Du, Zhang, Liu & Chen, 2007). The enzyme from microorganisms was named microbial transglutaminase (MTGase or MTG).
The MTGase from Streptomyces has been produced at an industrial scale by Ajinomoto Co., Inc., with the collaboration of Amano Enzyme, Inc.
The screening work and optimal conditions for the production of MTGases in a rotatory shaker have been described (Macedo, et.al., 2007; Macedo, et al., 2008). We recently isolated a new microbial strain from Brazilian soil samples, which is classified as Streptomyces sp. CBMAI 837. The objectives of this work were to purify and characterize the MTGase from the newly isolated Streptomyces sp. CBMAI 837. The scaled-up cultivation, downstream process and enzyme application are being investigated and are in the advanced stages of study (unpublished data).
MATERIAL AND METHODS
Material
Yeast extract, malt extract, peptone and agar were purchased from OXOID (Basingstone, Hants, England). KH2PO4, MgSO4.7H2O, glycerol, and glucose were
obtained from Synth (Detroit, MI., U.S.A). N-carbo-benzoxy-L-glycine and L-glutamic acid γ-monohydroxamate were purchased from Sigma (St. Louis, MO, U.S.A.). Soybean flour and the potato starch were obtained from Yasmin Food Industry (São Paulo, SP, Brazil) and Yoki Food Industry (São Bernardo do Campo, SP, Brazil), respectively. The low molecular mass protein calibration kit and the Sephadex G-75 resin were from GE Healthcare. Commercial transglutaminase (Activa® TG-BP) was provided from Ajinomoto Co., Inc.
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Microorganism and crude enzyme preparation (ver Anexo II para testes complementares)
Streptomyces sp. CBMAI 837, which was collected by our laboratory, was used throughout this study. The strain was cultivated in petri dishes containing ISP2 media (0.4% yeast extract, 1% malt extract, 0.4% glucose and 1.5% agar, pH 7.0 ± 0.2) for 7 days at 30°C, and two cylinders (6mm Ø) of the fully grown agar cultivation were removed from the petri dishes and preserved in cryotubes with a 10% glycerol solution at -80ºC. The content of one cryotube was inoculated into a 50 mL Erlenmeyer flask with 15 mL of fermentation medium and cultivated at 30°C for 5 days at 200 rpm in a rotatory shaker (Tecnal TE-421). The optimized fermentation medium consisted of 0.2% KH2PO4,0.1% MgSO4.7H2O, 2% soybean flour, 2% potato starch, 0.2% glucose, and 2%
peptone (Macedo et al., 2007). After five days, the culture broth was centrifuged at 10,000 x g for 15 min at 5°C to remove microorganisms, and the resulting supernatant solution was frozen and lyophilized. The enzymatic preparation was stored at -20 ºC.
Analytical methods
Transglutaminase activity
The transglutaminase activity was determined by hydroxamate formation with the specific substrate N-carbobenzoxy-L-glutaminyl glycine (N-CBZ-Gln-Gly) according to Folk & Cole, 1966, with some modifications. The reaction mixture, containing 200 µL of enzyme, 200 µL of 0.2 M citrate buffer pH 6.0, 25 µL of hydroxylamine, and 75 µL of 0.1 M N-carbobenzoxy-L- glutaminyl glycine, was incubated at 37°C for 1h and then stopped by adding an equal volume (500 µL) of 15% TCA containing 5% FeCl3. The
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activity was defined as the amount that causes the formation of one micromole of hydroxamic acid per minute at 37°C. A calibration curve was prepared using L-glutamic acid y-monohydroxamate.
Protein determination
The amount of protein was determined by the Bradford method (Bradford, 1976) with bovine serum albumin as the standard.
Purification of MTGase (ver Anexo III para estudos complementares)
All the steps were performed at 6°C, unless otherwise stated. Sephadex G-75 chromatography
A 1 g sample of crude enzyme powder (0.52 U of transglutaminase) was dissolved in 5 mL of 25 mM phosphate buffer, pH 6.0 and was applied on a Sephadex G-75 column (1.10 x 95 cm) pre-equilibrated with the same buffer. The column was washed with 0.1 M NaCl in the same buffer at a flow rate of 1.0 mL/15 min, and the active fractions were collected and pooled. Ten militers of the active pooled fraction obtained from the Sephadex G-75 column were applied to the same column under identical conditions, and the active fractions were pooled, lyophilized and stored at -20 ºC.
Sodium dodecylsulfate polyacrylamide gel electrophoresis
The purified enzyme was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Vertical Slab Mini-Protean Electrophoresis System Bio- Rad Laboratories, Hercules, CA, USA) according to Laemmli, (1970). A 12% separating gel was used. The proteins were stained with a 0.1% solution of Coomassie brilliant blue R-250.
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Biochemical characterization of MTGase
All procedures were carried out under the same conditions for samples of both the commercial MTGase and the pure and crude MTGase from Streptomyces sp. CBMAI 837. In order to compare the results obtained for each enzyme sample, the amount of enzyme powder in each trial was defined as the total measured protein in the sample. The total amount of protein present in each reaction was held constant (4 µg protein). All experiments were run in triplicate.
pH effect
The effect of pH on the MTGase activity was determined under standard assay conditions using a 50 mM sodium citrate buffer (pH 3.0-6.5), a 50 mM Tris-HCl buffer (pH 7.0-9.0) and a 50 mM Borax-NaOH buffer (pH 10.0). The enzyme activity was measured after 60 min at 37°C.
The effect of pH on MTGase stability was determined using the above mentioned buffer systems over the pH range 3.0-10.0. The enzyme solutions were incubated at the various pH values for 60 min at 25ºC without substrate. The remaining enzyme activity was then measured at 37ºC using N-CBZ-Gln-Gly in a sodium citrate buffer at pH 6.0. The relative activities were determined by using the maximal activity of the enzyme at a specific pH as 100%.
Temperature effect
The effect of temperature on MTGase activity was tested by assaying the activity at different temperatures (4ºC and over the range from 25°C to 70°C at pH 6.0) using the reaction mixtures indicated previously. The relative activities were determined by defining the maximal activity of the enzyme at a specific temperature as 100%. The
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enzyme’s thermal stability was determined by pre-incubating it at various temperatures for 30 min, and then placing the samples on ice immediately. The relative activities were then determined using the standard method described above.
The effect of metal ions
The effects of different metal ions were investigated by adding them (5 mM) to the reaction mixture and incubating at 37ºC for 60 min under the standard assay conditions. The relative activities were determined by using the activity of the enzyme in the absence of additives as 100%.
Kinetic parameters
Kinetic parameters were determined using the experimental system described above with varying amounts of the model substrate, N-CBZ-Gln-Gly (0-30 mM). The Michaelis constants (Km) and maximum velocities (Vmax) were determined from
Lineweaver-Burk plots.
RESULTS AND DISCUSSION
Purification of MTGase (ver anexo III para estudos complementares)
The enzyme was purified 5.0 fold with a yield of 17.7% after two steps of gel filtration chromatography.
The crude enzyme preparation was submitted to the initial purification step, the first chromatographic run on a Sephadex G-75 column (Fig.3.1), which resulted in a 2.3- fold purification, a yield of 48%, and a specific activity of 1.92 U/mg (Table 3.1). The usual first run on a CM-cellulose column described for MTGase purification in several cases (Soares et al., 2003; Yokoyama et al., 2004; Zhu et al., 1995; Klein et al., 1992; Ando et al., 1989; Cui et al., 2007; Ho, Leu, Hsieh & Jiang, 2000.) was avoided because
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the MTGase-ion exchange resins strongly affected the enzyme activity (data not shown). A further purification step was achieved by subjecting the pooled enzyme fraction from the first column step to a second run through the same Sephadex G-75 column (Fig. 3.1), which resulted in a 5.0-fold purification with a yield of 17.7% and a specific activity of 4.18 U/mg protein as compared to the crude extract (Table 3.1). Though both the purification fold and yield were lower after this step, protein purification was successfully achieved to SDS-PAGE electrophoretical homogeneity. From these results, the molecular mass of the MTGase from Streptoverticillium sp. CBMAI 837 was estimated to be about 45 kDa (Fig. 3.2). This result shows that the enzyme is heavier than that from Bacillus subtillis (29 kDa) (Suzuki et al., 2000), Streptomyces mobaraensis (37 kDa) (Ando et al., 1989), and Streptomyces hygroscopicus (38 kDa) (Cui et al., 2007), and approximately equal to those from Streptoverticillium sp S 8112 (40 kDa) (Ando et al., 1989) and Bacillus circulans (45 kDa) (Soares et al., 2003). However, it is smaller than other Ca+2-dependent tissue (Guinea pig liver, 90 kDa and tropical tilapia, 85 kDa) transglutaminase (Folk and Cole, 1966; Worratao & Yongsawatdigul, 2000).
This purification method proved to be rapid and practical, which are very important characteristics for downstream enzymatic methodology.
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Figure 3.1. Chromatography of the crude MTG from Streptomyces sp. CBMAI 837 on: (a) the first and (b) second run on the Sephadex G-75 column. The arrow indicates the fraction containing MTG activity.
Table 3.1. Summary of purification of MTG from Streptomyces sp. CBMAI 837 on
Sephadex G-75 column. Purification step MTG (U) Protein (mg) Specific activity (U/mg) Purification fold Yield (%)
Solution of lyophilized crude enzyme
extract (1.0 g/5 mL) 0.520 0.625 0.83 1 100 Sephadex G-75 (first run)
0.250 0.130 1.92 2.3 48 Sephadex G-75 (second run, lyophilized
MTG active fractions in solution, 150 mg/mL) 0.092 0.022 4.18 5.0 17.7 0 0,5 1 1,5 2 2,5 0 50 100 150 Elution volumn (mL) A b s o rb a n c e a t 2 8 0 n m -0,5 0 0,5 1 1,5 2 2,5 3 3,5 4 0 20 40 60 80 100 Elution volumn (mL) A b s o rb a n c e a t 2 8 0 n m 0 0,005 0,01 0,015 0,02 M T G a s e a c ti v it y ( U /m L ) 280 nm MTG activity (a) (b)
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Figure 3.2. SDS-PAGE of MTG on an acrylamide gel showing the (a) standard marker, (b) crude enzyme, and (c) MTG after the Sephadex G-75 second run.
Effect of pH on MTGase activity
The effect of pH on the activity of the different MTG samples was determined using the previously described reaction mixtures in the pH range from 3.0 to 10.0 at 37°C. The three different samples exhibited optimum activity for the catalytic reaction with N-CBZ-Gln-Gly in the 6.0-6.5 pH range. But the crude Streptomyces sp. enzyme and the commercial MTGase sample demonstrated a second maximum of activity at pH 10.0 (Fig. 3.3 (a)). This test was repeated several times, and control samples used to test for the possible degradation of the substrate at this alkaline pH developed no color. There is the possibility that two different transglutaminases were present in the unpurified sample. More experiments are needed to determine the properties of the enzyme that is active at pH 10.0. 94 KDa 67 KDa 43 KDa 30 KDa 20 KDa 14 KDa a b c
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The pH stability tests revealed that the purifyed and the commercial MTGase samples were stable from pH 5.0 to 8.0 (Fig. 3.3 (b)), but the commercial enzyme suffered a more gradual loss of activity above pH 8.0 than the other samples. The crude MTGase sample lost 20% of its enzymatic activity from pH 6.0-8.0. The optimum pH for these enzymes, about 6.0, was nearly the same as that reported for Streptomyces hygroscopicus (Cui et al., 2007). However, there are no literature reports of a second maximum in activity at pH 10.0. The activity of all three enzyme samples decreased gradually at alkaline pH values, but it decreased rapidly with increasingly acidic pH values.
The corresponding enzymes from mammals and fish have an optimum pH value of 8.0, while soybean MTGase has been reported to have an optimal pH value of 7.6 (Zhu et al., 1995; Worratao & Yongsawatdigul, 2000).
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(a) (b)
(c) (d)
Figure 3.3. Effect of pH on the (a) activity and (b) stability and the effect of temperature on (c) activity and (d) stability of: purified and crude MTG from
Streptomyces sp. CBMAI 837, and commercial MTG.
Effect of temperature on MTGase activity
The effects of temperatures on the activity of the MTG samples were studied by determining the activity of the samples at 4°C and at temperatures between 25-70°C at pH 6.0, under the conditions previously indicated. All the enzyme samples showed optimum activity in catalyzing the reaction of N-CBZ-Gln-Gly and hydroxylamine at 35- 40°C. Almost no enzyme activity was detected at 60°C for either the crude or purified
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MTG from Streptomyces sp., but the commercial sample still displayed some residual activity (Fig. 3.3 (c)). The temperatures required for the optimum activity of these MTG enzymes are very similar to the optimum temperatures reported for Streptomyces hygroscopicus and Streptoverticillium ladakanum (Cui et al., 2007; Ho et al., 2000). However, the MGT enzyme from Streptomyces sp. is completely different from Bacillus subtilis transglutaminases, which have optimal temperatures of 60°C (Suzuki et al., 2000).
The thermal stability of the enzymes was investigated between 4 and 70°C, and on average, they retained about 80% of their activity in the temperature range from 4 to 45°C after a 30 min incubation at pH 6.0. The activity of the purified enzyme was nearly zero and for the other two samples decreased to 10%, after incubation at 60°C (Fig.3.3 (d)).
Effect of different inhibitors and metal ions on MTGase activity
The relative activity of the transglutaminases was investigated in the presence of several metal ions and EDTA (ethylenediaminetetraacetic acid), which were added in different concentrations according to the method described above. As shown in Table 3.2, all three MTG samples were strongly inhibited by Cu2+ and Hg2+. These metal ions are known to react preferentially with thiol groups, and the strong inhibition of the enzyme by these ions suggests that this enzyme contains a thiol group in its active site, similar to other TGases from both vertebrate tissues and microorganisms (Soares et al., 2003; Ando et al., 1989; Ho et al., 2000). On the other hand, the presence of K+, Ba2+, and Co2+ led to an increase in the activity of the crude and purified extracts from Streptomyces sp. MTG. However, these same metal ions decreased the MTG activity of the commercial enzyme.
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These differences may be significant in some industrial processes, and this demonstrates the importance of characterizing new enzymes. The presence of Ca2+ increased the MTG activity in all of the samples, but only by 10-19%, which suggests that these enzymes are calcium independent.
Under the same incubation conditions and metal ion concentrations, the enzyme from Streptomyces hygroscopicus was strongly inhibited by Zn2+, Cu2+,Hg2+, Pb2+ and Fe3+ (Cui et al., 2007).
The effect of EDTA on the activity of the MTG samples is shown in Table 3.3. The different MTG samples reacted very differently to the presence of EDTA. The commercial enzyme was unaffected by the presence of EDTA at all of the concentrations tested. The purified MTG extract from Streptomyces sp. showed decreased activity as the EDTA concentration increased. There was a 30% loss of enzyme activity when the EDTA concentration was increased from 0 to 5 mM. However, the crude MTG extract from Streptomyces sp. did not show a difference in activity until the EDTA concentration reached 5 mM, at which point the activity decreased by about 50%.
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Table 3.2. The influence of various metal ions on the activity of purified and crude
Streptomyces sp. CBMAI 837 transglutaminase and commercial transglutaminase. MTG relative activity (%)
Metal ion (5mM) Crude enzyme Pure enzyme Commercial enzyme
Co2+ 132 118 85 Cu2+ 21 21 21 K+ 124 126 89 Ca2+ 118 112 119 Fe3+ 128 97 88 Zn2+ 118 74 138 Ba2+ 121 123 106 Hg2+ 9 11 11 Mg2+ 114 83 101 Na+ 108 118 107 Mn2+ 99 115 88 None 100 100 100
Table 3.3. The effect of EDTA on the activity of purified and crude Streptomyces sp. transglutaminase and commercial transglutaminase.
MTG relative activity (%)
EDTA (mM) Crude enzyme Pure enzyme Commercial enzyme
0 100 100 100
0.1 111 105 108
0.5 98 83 105
1 100 96 97
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Determination of kinetic parameters
The effect of substrate concentration on the velocity of the enzymatic reaction was determined at pH 6.0 and 37°C. The crude enzyme preparation from Streptomyces sp. presented a Km of 6.52 mM and a Vmax of 1.35 U/mL, while the purified enzyme
presented a Km of 6.37 mM and a Vmax of 1.7 U/mL for the reaction with N-CBZ-Gln-
Gly. These values were derived from the corresponding Lineweaver-Burk plots (Fig.3.4).
(a) (b)
Figure 3.4. Determination of kinetic parameters of the purified and crude MTG from Streptomyces sp. CBMAI 837 (a) Kinetic studies, (b) Lineweaver-Burk plot.
Under the same conditions, the transglutaminase from Streptomyces hygroscopicus, studied by Cui et. al., 2007, presented a Km of 54.69 mM and a Vmax of
1.28U/mL. And the Streptomyces mobaraense transglutaminase, studied by Gerber et al., 1994, showed a Km of 12.2 mM. The lower values of Km presented by both forms
of the transglutaminase from Streptomyces sp. CBMAI 837, indicates a higher affinity of this enzyme for the substrate N-CBZ-Gln-Gly.
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CONCLUSIONS
This paper reports the purification and characterization of transglutaminase from a newly isolated Streptomyces sp. CBMAI 837. The MTG was purified after two fast and simple steps on the same column, and the molecular mass of the enzyme was estimated to be 45 kDa. In terms of optimum activity and stability over a range of pH and temperature, the new transglutaminase performed in a manner very similar to the commercial transglutaminase. All of the samples exhibited optimal activity in the pH range 6.0-6.5 and at 35-40°C. A second maximum in activity was observed at pH 10.0 for both the crude Streptomyces sp. enzyme and the commercial enzyme. This interesting fact has not been previously reported in the literature. The crude preparation may contain two different transglutaminase.
All of the enzymes preparations tested were stable over a broad pH range (4.5- 8.0) and up to 45°C. This biochemical characterization revealed that MTG from Streptomyces sp. CBMAI 837 had a pH response similar to the commercially available enzyme, which is good for food processing, because it is stable over a broad pH range with optimum activity near neutral pH. The catalytic activities of all the MTG samples were independent of Ca+2, but they were enhanced in the presence of K+, Ba2+, and Co2+ and inhibited by Cu2+ and Hg2+, which suggests a thiol group in their active sites. The purified enzyme demonstrated a Km of 6.37 mM and a Vmax of 1.7 U/mL, while the crude
enzyme exhibited a Km of 6.52 mM and a Vmax of 1.35 U/mL.
The transglutaminase from Streptomyces sp. CBMAI 837 is a good candidate for applications in the food industry. However, additional work to increase the activity of the